Laser ablation cell

ABSTRACT

A laser ablation cell ( 1 ) comprises a flow channel ( 11 ) having an essentially constant cross-sectional area so as to ensure a strictly laminar flow in the flow channel. A sample chamber ( 21 ) is provided adjacent to a lateral opening ( 14 ) of the flow channel. A laser beam ( 41 ) enters the sample chamber ( 21 ) through a lateral window ( 16 ) and impinges on a surface ( 24 ) of a sample ( 23 ) to ablate material from the sample. The sample may be positioned in such a distance from the flow channel that the laser-generated aerosol mass distribution has its center within the flow channel. This leads to short aerosol washout times. The laser ablation cell is particularly well suited for aerosol generation in inductively coupled plasma mass spectrometry (ICPMS), including imaging applications.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/223,606, filed on Mar. 24, 2014, which is a continuation ofInternational PCT Application No. PCT/EP2013/056115, filed Mar. 22,2013, the entire contents of which are hereby incorporated herein byreference.

TECHNICAL FIELD

The present invention relates to a laser ablation cell, to an ablationapparatus and an inductively coupled plasma (ICP) ion source employingsuch a laser ablation cell, and to a method of using such a laserablation cell, for example for the imaging of biological material.

BACKGROUND

Inductively coupled plasma mass spectrometry (ICPMS) provides accuratequantitative information on major, minor, trace, and ultra-traceelements of industrial, geological, environmental, and biologicalsamples. In ICPMS, an aerosol sample is carried by a carrier gas streamto a so-called ICP torch. In this torch, the gas is subjected to intensehigh-frequency electromagnetic fields, which lead to the formation of aplasma by induction. The ions from the plasma are then extracted into amass spectrometer, where they are separated on the basis of theirmass-to-charge ratios.

ICPMS can be coupled with laser ablation (LA) to ablate material from asolid sample so as to create the aerosol required for ICP. Ablation maybe carried out directly in the ICP torch, or the sample may be placed inan external laser ablation cell upstream of the ICP torch, and theaerosol created by laser ablation is transported to the ICP torch by thecarrier gas stream. For example, reference 1 demonstrated a laserablation cell (the so-called HEAD cell) for which the aerosol ejectiondirection is parallel to that of the carrier gas. Another laser ablationcell design based on a similar principle is demonstrated in reference 2.

Since the first half of 1990s, attempts have been made to uselaser-ablation ICPMS (LA-ICPMS) as a chemical imaging tool by scanningthe laser spot over the sample surface. Many studies have demonstratedthe potential imaging capabilities of LA-ICPMS based on a considerablevariety of hard and soft samples. Most of these studies showed aneffective spatial resolution of approximately 5-100 μm. AlthoughLA-ICP-MS offers highly multiplexed quantitative analysis of antigenexpression in single cells, it currently lacks the resolution necessaryfor the imaging of single cells within tissue samples.

However, some applications, such as diagnostic analysis of tissuesections, requires higher spatial resolution, e.g. to visualizecell-to-cell variability. The effective spatial resolution is determinedby the laser spot size convoluted with the system dispersion. The systemdispersion is in turn often dominated by a compromise between theaerosol washout time after each laser shot and the scanning speed. Thelonger the washout time, the more overlap will occur between signalsoriginating from neighboring sample spots if the scanning speed is keptfixed. Therefore, aerosol washout time often is one of the key limitingfactors for improving resolution without increasing total scan time.

The fastest washout time can be achieved by in-torch ablation, resultingin single shot signal durations of a few milliseconds. However, in-torchablation is limited to very small samples, and scanning of the laserspot is very difficult to realize with in-torch ablation. Therefore, forimaging applications, external laser ablation cells are generallyemployed. However, even with the best known cell designs, washout timesare often on the scale of seconds, and short washout times under 100milliseconds are hard to achieve.

It is an object of the invention to provide further and improved laserablation cells, ablation apparatus incorporating such cells (forexample, linked to an ICP-MS), which have an application in techniquesfor imaging of biological material, such as tissue samples, monolayersof cells and biofilms, and in particular to adapt LA-ICP-MS for use as asingle-cell imaging technique.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a laser ablation cellthat has the potential of achieving short aerosol washout times.

Accordingly, a laser ablation cell is provided, comprising a flowchannel having an inlet for feeding a carrier gas to the flow channeland having an outlet. A lateral opening is provided in a first wallportion of the flow channel, and a lateral window is disposed in asecond wall portion of the flow channel opposite of the lateral opening.A sample chamber is provided adjacent to the lateral opening. The samplechamber is configured to receive a sample in a manner to enable a laserbeam to enter the sample chamber through the lateral window and thelateral opening and to impinge on a surface of the sample, so as toablate material from the sample surface and to create an aerosol. Thesample chamber has an inlet for feeding a sheath gas to the samplechamber.

The inlet and outlet of the flow channel may be connected to tubinghaving essentially the same cross-sectional area as the flow channelitself. In this manner, the flow channel essentially acts like a singlepiece of tubing. The ablation cell of the present invention maytherefore be considered to have a “tube cell” design. By minimizingvariations of the cross-sectional area (and preferably also of thecross-sectional shape) of the flow channel, this “tube cell” designallows maintaining an essentially laminar flow pattern in the flowchannel, avoiding turbulences as far as possible. Furthermore, thedesign allows positioning the sample sufficiently close to the flowchannel that a major proportion of the laser-induced aerosol plume isintroduced directly into the flow of the carrier gas. These measuressignificantly reduce dispersion. In practice, the present cell designallows reducing the washout time to below 30 ms (full width at 1%maximum) and minimizing the tailing of the sample washout. Thisimprovement is observed for elements across the entire atomic massrange. Thus the invention provides a tube cell laser ablation cell. Theinvention also provides an ablation cell with a wash out time of lessthan 90 ms, for example less than 30 ms.

The flow channel preferably has a cross-sectional area that isessentially constant or at most varies weakly. In particular, preferablythe cross-sectional area of the flow channel is essentially constant inthe vicinity of the lateral opening. The cross-sectional area of theflow channel may be regarded to vary at most weakly if any variations incross section do not significantly disturb laminar flow. In particular,the cross-sectional area may be considered to vary at most weakly if thevariation of the average diameter of the flow channel is less than 1.5mm per 1 mm length along the tube axis, preferably less than 0.5 mm per1 mm length along the tube axis, more preferably less than 0.2 mm per 1mm length along the tube axis for any cross-sectional plane along theflow channel. It is preferred that the flow channel does not form apronounced constriction at the lateral opening so as to avoid pronouncedsuction effects, as in a Venturi tube, and that the flow channel doesnot widen up significantly in the vicinity of the lateral opening so asto avoid that the carrier gas is pushed into the sample chamber by theresulting positive pressure difference.

In absolute numbers, the cross-sectional area may take a wide range ofvalues, depending on laser spot size and laser energy. The mean diameterof the flow channel (calculated as d=2√{square root over (A/π)}, where Ais the cross-sectional area) may range, e.g., from 50 micrometers to 5millimeters, preferably from 200 micrometers to 5 millimeters.

The angle between the inlet and the outlet of the flow channel ispreferably at least 160° (to be precise, between 160° and 200°), morepreferably at least 170° (to be precise, between 170° and 190°). Inother words, the flow channel is preferably essentially straight or bentby not more than 20° or better not more than 10° in any direction. Anarbitrary direction of the sheath gas inlet relative to the direction ofthe flow channel may be chosen. Preferably, the sheath gas inlet extendsperpendicular to the transversal direction.

The flow channel and the sample chamber are separated by a separatingwall, which forms the first wall portion of the flow channel in whichthe lateral opening is arranged. In order to allow the sample to bepositioned sufficiently close to the flow channel, the separating wallpreferably has a minimum thickness of less than 500 micrometers, morepreferably less than 200 micrometers. It should be noted that thethickness of the separating wall may vary along the circumference of theflow channel; the separating wall will normally have its smallestthickness immediately adjacent to the opening between the tube and thesample chamber, and the thickness will increase away from the opening ina plane perpendicular to the tube axis. The thickness may further varyalong the length of the flow channel.

In order to minimize flow disturbances induced by the lateral opening,the cross-sectional area of this opening should be kept small. On theother hand, it may be desirable to make the opening sufficiently largeto enable the laser beam to be scanned over the sample surface withoutmoving the sample relative to the opening. As a compromise, thecross-sectional area of the lateral opening is preferably not more thanabout 20 mm², more preferably not more than about 7 mm². Expressedrelative to the cross-sectional area of the flow channel, the ratio ofthe cross-sectional areas of the opening and the flow channel ispreferably not more than about 5, more preferably not more than about 3,most preferably not more than about 1. In order to enable a laser beamto pass through the lateral opening, the lateral opening shouldpreferably have a cross-sectional area of at least about 0.01 mm². Thewidth of the lateral opening transverse to the flow channel ispreferably less than 80% of the mean diameter of the flow channel, andmore preferably less than 50% of the mean diameter of the flow channel.The length of the lateral opening in the direction of the flow channelis preferably not more than five times the mean diameter of the flowchannel and more preferably not more than three times or even 1.5 timesthe diameter of the flow channel.

In order to enable easy sample exchange, the laser ablation cellpreferably has a two-part design, comprising a first cell part (in thefollowing referred to as a “cell top”) that houses the flow channel anda second cell part (in the following referred to as a “cell bottom”)that forms the sample chamber. The cell bottom is preferably removablefrom the cell top for exchanging the sample. The cell bottom ispreferably open towards the cell top, i.e., the separating wall betweenthe sample chamber and the flow channel is preferably formed by the celltop rather than by the cell bottom. The terms “top” and “bottom” are tobe understood as not defining an absolute orientation of these parts;these terms are only used to better distinguish between the differentcell parts, and the laser ablation cell may as well be used in aninverted orientation where the cell top is pointing towards the floorand the cell bottom is pointing towards the ceiling.

The invention further relates to a complete ablation apparatuscomprising an ablation cell as described above. The ablation apparatusfurther comprises a laser, in particular, a UV laser, for shooting alaser beam through the lateral window and the lateral opening and ontothe sample surface, and a positioning device for changing the relativeposition between the sample and the laser beam. The positioning devicemay comprise, e.g., any of the following: an x-y or x-y-z stage formoving the entire laser ablation cell relative to the laser; an x-y orx-y-z stage for moving the sample within the laser ablation cell whilekeeping the relative position between the ablation cell and the laserfixed; a beam deflector for deflecting the laser beam while keeping therelative position between the ablation cell and the laser fixed; etc.The positioning device may be employed to scan the laser beam over thesample surface. The resulting aerosol may subsequently be analyzed withrespect to its elemental or isotopic composition, e.g., by ICPMS. Inthis manner, the sample surface may be imaged according to its elementalor isotopic composition. However, the present invention is not limitedto the use of the ablation cell in conjunction with ICPMS imaging andmay also be employed in other methods in which short aerosol pulses arerequired.

The invention further provides an ICP ion source comprising an ablationcell as described above. The ICP source further comprises an ICP torchconnected to the outlet of the ablation cell, and tubing connecting saidablation cell to the ICP torch. Preferably the tubing has across-sectional area that is essentially identical to thecross-sectional area of the flow channel of the ablation cell or changesonly weakly as compared to the cross-sectional area of the flow channel,so as to maintain a laminar flow with minimum dispersion over the entirelength of the tubing.

The invention also encompasses an ICPMS system comprising such an ICPion source and a mass analyzer coupled to the ion source. The massanalyzer may, e.g., be a quadrupole mass analyzer, a time-of-flight(TOF) mass analyzer, or a sector field mass analyzer, in particular, aMattauch-Herzog mass analyzer. However, the invention is not restrictedto any particular type of mass analyzer.

The invention further provides a method of operating an ablation cell asdescribed above. The method comprises, not necessarily in the givenorder:

placing a sample in the sample chamber such that a surface of the samplefaces the lateral opening;

feeding a carrier gas to the inlet of the flow channel;

feeding a sheath gas to the inlet of the sample chamber; and

ablating material from the surface by shooting a pulsed laser beamthrough the lateral window and the lateral opening and onto saidsurface.

The direction of the laser beam, the orientation of the lateral windowand the lateral opening, and consequently the orientation of the samplemay be arbitrary in space. For instance, the laser beam may be directedupwards, downwards, sideway etc., and the sample surface may be orientedin any orientation that allows the laser beam to reach the samplesurface.

Each laser pulse will cause a quasi-instantaneous laser-generatedaerosol mass distribution (“plume”). Here, “quasi-instantaneous” means atime scale that is much shorter than the time scale of mass transport bythe carrier gas stream and the sheath gas stream. The laser-generatedaerosol mass distribution is caused by the action of the laser pulsealone, neglecting the normal gas flow of the carrier and sheath gases.This mass distribution is established within less than 1 millisecondafter the first interaction of the laser pulse with the sample. Thesample is preferably positioned at such a distance from the flow channelthat the quasi-instantaneous laser-generated aerosol mass distributionhas its center within the flow channel, between the lateral opening andthe lateral window. The center of the mass distribution is defined inthe usual manner, in the same way as the center-of-mass of a rigid body,integrating over the entire aerosol plume. In this manner, the majorityof the aerosol plume is directly injected into the stream of the carriergas and may be transported away by the stream of the carrier gas withminimum dispersion.

The optimum distance between the sample surface and the center axis ofthe flow channel will depend on the type of laser, the energy of thelaser beam, the type of carrier and sheath gases, and the flow rates ofthe gases. For instance, for a standard ArF excimer laser with pulses inthe nanosecond range, argon as carrier gas at a flow rate of 1.1 L/min,and helium as sheath gas at a flow rate of 0.6 L/min, a distance ofabout 2 mm has turned out to be optimal. In more general terms, thesample should preferably be positioned in such a manner that the surfaceof the sample has a distance from the center axis of the flow channel inthe range of 0.5 millimeters to 4.5 millimeters for laser pulses in therange of 50 femtoseconds to 50 nanoseconds.

In consequence, the optimum distance between the sample surface and theseparating wall that separates the sample chamber from the flow channelwill depend on various parameters. This distance may range from lessthan 50 micrometers to 1 millimeter or more. The distance should belarge enough to allow the sheath gas to flow along the surface of thesample and through the lateral opening into the flow channel.

The sheath gas fulfills at least three tasks: it flushes the aerosol inaxis with the aerosol injection direction, which helps the uptake of theaerosol particles in the carrier gas stream; it forms a “protectionregion” above the sample surface and ensures that the ablation iscarried out under a controlled atmosphere; and it increases the flowspeed in the flow channel. Preferably the viscosity of the sheath gas islower than the viscosity of the primary carrier gas. This helps toconfine the aerosol in the center of the flow channel and to minimizethe aerosol dispersion downstream from the ablation cell. In particular,the carrier gas is preferably argon (Ar). Argon is particularlywell-suited for stopping the aerosol expansion before it reaches thewalls of the flow channel, and it is also required for an improvedinstrumental sensitivity in most of the Ar gas based ICP. The sheath gasis preferably helium (He). However, the sheath gas may be replaced by orcontain other gases, e.g., hydrogen, nitrogen, or water vapor.Preferably the main proportion of the sheath gas is helium, e.g., atleast 50% by volume. At 25° C., Ar has a viscosity of 22.6 μPas, whereasHe has a viscosity of 19.8 μPas.

The optimum flow rates of the carrier gas and the sheath gas will dependon a variety of factors, first of all on geometry, in particular, on thecross-sectional area of the flow channel. Secondly, they will depend onthe geometry of the lateral opening. However, it is preferred that thevolume flow rate of the sheath gas is smaller than the volume flow rateof the carrier gas, in particular, 0.3 to 1.0 times the volume flow rateof the carrier gas. The flow rate of the sheath gas may be adjusted tohelp to inject the center of the aerosol plume close to the center ofthe flow channel.

The method of the present invention is particularly suited for chemicalimaging. To this end, the above method may comprise scanning the laserbeam over the surface and analyzing the resulting aerosol to obtain achemical image of the sample surface. Analysis may be carried out bymass spectrometry, in particular, by ICPMS, but may also be carried outby any other suitable method.

The method is well suited for the investigation of biological samples,in particular, of tissue samples of human or animal tissue. However, themethod is not limited to biological samples and may as well be appliedto other kinds of samples. When performed on biological samples, themethod of the invention may be performed at a subcellular level, at acellular level, or at lower resolution, wherein individual cells withina tissue are not individually resolved. In certain embodiments, themethod of the invention uses the specific laser ablation cell disclosedherein. Thus the invention provides the use of an ablation cell with awash out time of less than 100 ms for analysing biological samples.

The invention therefore also provides a method of imaging biologicalsamples, such as a tissue sample, comprising (i) labelling at least oneof a plurality of different target molecules in the tissue sample withat least one different labelling atom to provide a labelled tissuesample, wherein the labels can be distinguished by LA-ICP-MS to identifyeach of the at least one different target molecules, (ii) subjectingmultiple cells of the labelled biological sample to laser ablation atmultiple known locations, to form a plurality of plumes; and (iii)subjecting plumes to inductively coupled plasma mass spectrometry,whereby detection of labelling atoms in the plumes permits constructionof an image of the biological sample.

In some embodiments, the at least one of a plurality of different targetmolecules is labelled with at least one different specific labellingconstruct (e.g. an antibody or nucleic acid or the like that bindsspecifically to the target molecule), wherein each different labellingconstruct is attached to a lanthanide labelling atom, and the laserablation is at a resolution of the cellular level or better.

In some embodiments, each of the at least one of a plurality ofdifferent target molecules is labelled with at least one differentspecific labelling constructs, wherein each different labellingconstruct is attached to a lanthanide labelling atom, and the laserablation is performed with a laser spot size of 25 μm or less.

In some embodiments, at least three different target molecules arelabelled with at least three different labelling atoms, and the laserablation is at a resolution of the cellular level or better.

In some embodiments, at least three different target molecules arelabelled with at least three different labelling atoms, and the laserablation is performed with a laser spot size of 25 μm or less.

In some embodiments, the at least one of a plurality of different targetmolecules is labelled with at least one different labelling atom, andthe laser ablation is at a resolution of the sub-cellular level.

In some embodiments, the at least one of a plurality of different targetmolecules is labelled with at least one different labelling atom, andthe laser ablation is performed with a laser spot size of 4 μm or less.

In some embodiments, the at least one of a plurality of different targetmolecules is labelled with at least one different labelling atom, andthe inductively coupled plasma mass spectrometry uses a time-of-flightdetector.

The invention also provides methods in which the analysis of biologicalsamples is a step in methods of diagnosis, treatment, or monitoring ofpatients suffering from a disease.

These methods can use the ablation cell, ablation apparatus, ICP ionsource or system as disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention are described in the followingwith reference to the drawings, which are for the purpose ofillustrating the present preferred embodiments of the invention and notfor the purpose of limiting the same. In the drawings,

FIG. 1 shows a schematic sketch (not to scale) of a laser ablation cellaccording to the present invention in perspective view;

FIG. 2 shows a schematic sketch (not to scale) of the laser ablationcell of FIG. 1 in a central longitudinal section;

FIG. 3 shows a schematic illustration (not to scale) of thelaser-generated plume in the laser ablation cell;

FIG. 4 illustrates, in a schematic manner, simulation results for thegas flow in the flow channel of the laser ablation cell; part (a) showsthe mixing distribution pattern between He and Ar, where the ablatedaerosol is located at the mixing interface above the lateral opening,the degree of mixing being indicated in gray scale, white representingthe highest degree of mixture; part (b) shows the simulated gas flowvelocity pattern, flow velocity being indicated in gray scale, whiterepresenting the highest flow velocity;

FIG. 5 illustrates, in a highly schematic manner, a complete LA-ICPMSsystem employing the laser ablation cell of FIG. 1;

FIG. 6 shows diagrams illustrating the results of optimizations of theperformance of the laser ablation cell by varying (a) the gap spacebetween the sample surface and the lateral opening; (b) the carrier gas(Ar) flow rate; and (c) the sheath gas (He) flow rate; all diagrams showpeak width normalized to peak area based on full width at 1% maximumcriterion;

FIG. 7 shows diagrams illustrating the performance of the laser ablationcell as demonstrated by ²⁷Al intensity in a mass spectrometer at variousrepetition rates: (a) transient signal for a repetition rate ofapproximately 1 Hz; (b) enlarged view of the bracketed portion of part(a); (c) transient signal for a repetition rate of approximately 10 Hz;(d) transient signal for a repetition rate of approximately 30 Hz;

FIG. 8 shows diagrams illustrating the characterization of the laserablation cell for various isotopes; (a) peak width; (b) abundancenormalized sensitivity calculated from peak area;

FIG. 9 shows images obtained for a Pt coated test pattern with an Aufilm in the form of letters “ETH” and an overlaid Ag film in the form ofletters “PSI”; imaging was carried out by (a) optical microscopy; (b)scanning electron microscopy; (c) and (d) LA-ICP-Quadrupole-MS employingthe laser ablation cell of FIG. 1; and

FIG. 10 illustrates an image of human epidermal growth factor receptor 2(HER2) distribution in a thin section cut of a breast cancer tissueobtained by LA-ICP-Single-Detector-Sector-Field-MS.

DESCRIPTION OF PREFERRED EMBODIMENTS

Laser Ablation Cell

FIGS. 1 and 2 illustrate, in a schematic manner, a laser ablation cell1, in the following also called a “tube cell”, according to an exemplaryembodiment of the present invention. The ablation cell 1 comprises twoparts: a first part or cell top 10 and a second part or cell bottom 20.A tubular flow channel 11 is formed in the cell top 10 and extends froma carrier gas inlet 12 to a mixed-gas outlet 13. In a bottom wallportion 15 of the cell top 10, a lateral opening 14 is formed. In a topwall portion 17 of the cell top 10, a transversal hole is formed andclosed by an UV transparent silicon window 16. In the cell bottom 20, asample chamber 21 is provided. A sheath gas inlet 22 leads to the samplechamber 21. Whereas the sheath gas inlet 22 is shown as extending(anti-)parallel to the flow channel 11, an arbitrary direction of thesheath gas inlet may be chosen. Preferably, the sheath gas inlet extendsperpendicular to the transversal direction. A sample 23 is placed in thesample chamber 21, and the cell bottom 20 is mounted to the cell top 10such that the top surface 24 of the sample 23 is situated below thelateral opening 14.

For operating the ablation cell, a carrier gas G1 is fed to the inlet 12of the flow channel 11, and a sheath gas G2 is fed to the inlet 22 ofthe sample chamber 21. A UV laser beam 41 enters the window 16,traverses the flow channel 11, exits the flow channel 11 through thelateral opening 14 and impinges on the top surface 24 of the sample 23.

Each laser pulse generates an aerosol plume 25, as schematicallyillustrated in FIG. 3. This plume is the direct result of the action ofthe laser pulse; the initial mass distribution in the plume immediatelyafter the end of the laser pulse is influenced only very little by thestreams of the carrier gas G1 and the sheath gas G2. The design of thelaser ablation cell 1 allows placing the center of the laser-generatedaerosol mass distribution right in the flow channel, without the need offirst transporting the aerosol to the flow channel by the sheath gasstream. The carrier gas G1 and sheath gas G2 then carry away the aerosoltowards the outlet 13, where they exit the ablation cell as a mixed-gasstream G3.

As the carrier gas G1, argon (Ar) is preferred. As the sheath gas,preferably helium (He) is chosen. Ar is beneficial for stopping theaerosol expansion typically occurring in pure He atmospheres. Adding Hefrom the sample container has three advantages: a) this setup flushesthe aerosol in axis with the aerosol injection direction, which helpsthe uptake of the particles; b) He gas forms a ‘protection’ region abovethe sample surface and assures that the ablation is conducted under Heatmosphere; c) mixing Ar and He already in the tube cell not only avoidsthe need of a dispersive gas adapter (Ar/He mixing bulb), but alsoincreases the flow speed and gas viscosity comparing to normal setupusing only He as carrier gas, hence decreases the aerosol dispersion.

FIG. 4 shows results of computational fluid dynamics simulations carriedout using the ANSYS CFX 12 software package (ANSYS Inc., Berlin,Germany). A shear stress transport model for turbulence was consideredin the simulation. Simulations were carried out for the followingparameters: Length of the lateral opening: L=4.5 mm; width of thelateral opening: 1.5 mm; minimum thickness of the bottom wall portion:w=50 micrometers; total length of the flow channel: 50 mm; diameter ofthe cylindrical sample chamber: 23 mm; distance between the top surface24 of the sample 23 and the bottom wall portion 15: d=350 micrometers.Ar flow at the inlet was set to a constant speed of 2.6 m/s (1.1 L/min),while He was simulated using 1.4 m/s (0.6 L/min).

The mixture distribution of the two inlet gases is shown in FIG. 4 (a).The mixing of the two gases is indicated in gray scale, white being thehighest degree of mixture. A sharp interface at the lateral opening 14is formed by the He flow entering into the Ar flow. Helium significantlydominates the opening region and forms together with Argon a boundarylayer. Due to the least degree of mixing of the two gases at the lateralopening 14, and the previous results indicating that laser ablatedaerosol penetrates easily in He but not in Ar, it can be assumed thatthe aerosol is not diffusing into the Argon atmosphere, is therefore notreaching the entire cross section of the flow channel, and remains verydense. The initially very sharp interface widens within a fewmillimeters downstream from the opening. By varying the combination ofthe inlet gas flows, the height of the boundary layer and accordinglythe height of the ablated aerosol can be controlled.

The simulated gas flow speed distribution is shown in FIG. 4 (b). The Arinlet flow upstream of the lateral opening 14 represents a typicallaminar flow distribution in the flow channel, being the fastest flow inthe center of the tube, and decreasing gradually towards the tube wall.Simulating the emergence of the two gases showed no significantturbulence flow. Nevertheless, the calculated Reynolds number (˜2000) isclose to the transition from laminar to turbulent flow (2300˜4000).However, using a turbulent model indicated a stringent laminar flow.Therefore it can be concluded that due to the absence of turbulences, adefined stopping distance for the laser aerosol close to the center ofthe tube cell which is matching the highest gas velocity, low aerosoldispersion should be achieved.

FIG. 5 schematically illustrates a complete LA-ICPMS system. The laserbeam is generated by a laser 40. The laser ablation cell 1 is mounted onan X-Y-Z stage 5 so as to be able to change the position of the samplerelative to the laser beam. The outlet 13 of the laser ablation cell 1is connected to an ICP torch 6 by tubing 61. The tubing 61 hasessentially the same inner diameter as the flow channel 11 of the laserablation cell 1 so as to ensure laminar flow of the outlet gas G3. TheICP torch generates a plasma source by operation of an RF coil 62; it isconstructed in the usual manner ICP torches are well known in the artand do not require further explanations. The ICP torch is connected to amass analyzer 7 via an ICP source 71. The mass analyzer may be aquadrupole mass analyzer, a time-of-flight (TOF) mass analyzer, a sectormass analyzer etc.

Of course, many modifications of the laser ablation cell and theLA-ICPMS setup are possible without leaving the scope of the presentinvention. In particular, the present invention is not limited to aparticular choice of materials for the laser ablation cell, to aparticular geometry or size of the sample chamber, to a particulargeometry, length, and diameter of the flow channel in the ablation cell,to a particular geometry and size of the lateral opening in the ablationcell, to a particular window size or material, to a particular type oflaser for ablation, to particular gas types introduced into the ablationcell, etc.

LA-ICP-MS Applications

ICP-MS has been used for single-cell analysis, but only for cells insuspension [3]. Laser ablation (LA) coupled to ICP-MS has now beenadapted so that this technique is suitable for single-cell imaging inbiological samples, such as in tissue samples. Although tissue imagingvia LA-ICP-MS has previously been reported, the disclosed procedureshave not achieved high spatial resolution. For instance, LA-ICP-MS hasbeen used to obtain images of brain sections [4] and of cancer tissue[5, 6] but single-cell or subcellular resolution was not achieved. Inall cases the resolution was limited by the spot size of the ablatinglaser and/or by overlap/mixing between the analysis of material releasedin consecutive ablations. For instance, a spot size of 40 μm [4] cannotablate material with a resolution which is high enough for single-cellimaging in a tissue sample. Similarly, ablation at a frequency of 10 Hzwill lead to overlapping signals if the ablated material cannot betransported to the ICP-MS in less than 100 ms; for example the ablationcell used in reference 5 with this ablation frequency had a washout timewell in excess of 1 s (which the authors did not note).

An LA-ICP-MS system, which overcomes these drawbacks and is able toachieve sub-cellular resolution, is provided herein, thereby makingavailable for the first time LA-ICP-MS techniques suitable forsingle-cell imaging of biological samples such as tissues. Thesetechniques include the methods described below.

The invention therefore provides a method of imaging a biologicalsample, such as a tissue sample, comprising labelling at least one of aplurality of different target molecules in the biological sample with atleast one different labelling atom, wherein each target is now labelledin a fashion it can be specifically identified, to provide a labelledbiological sample, subjecting multiple cells of the labelled biologicalsample to laser ablation at multiple known locations, to form aplurality of plumes; and subjecting plumes to inductively coupled plasmamass spectrometry, whereby detection of labelling atoms in the plumespermits construction of an image of the biological sample.

In some embodiments, the at least one of a plurality of different targetmolecules is labelled with at least one different specific labellingconstruct (e.g. an antibody or nucleic acid or the like that bindsspecifically to the target molecule), wherein each different labellingconstruct is attached to a lanthanide labelling atom, and the laserablation is at a resolution of the cellular level or better.

In some embodiments, the at least one of a plurality of different targetmolecules is labelled with at least one different specific labellingconstructs, wherein each different labelling construct is attached to alanthanide labelling atom, and the laser ablation is performed with alaser spot size of 25 μm or less.

In some embodiments, at least three different target molecules arelabelled with at least three different labelling atoms, and the laserablation is at a resolution of the cellular level or better.

In some embodiments, at least three different target molecules arelabelled with at least three different labelling atoms, and the laserablation is performed with a laser spot size of 25 μm or less.

In some embodiments, the at least one of a plurality of different targetmolecules is labelled with at least one different labelling atom, andthe laser ablation is at a resolution of the sub-cellular level.

In some embodiments, the at least one of a plurality of different targetmolecules is labelled with at least one different labelling atom, andthe laser ablation is performed with a laser spot size of 4 μm or less.

In some embodiments, the at least one of a plurality of different targetmolecules is labelled with at least one different labelling atom, andthe inductively coupled plasma mass spectrometry uses a time-of-flightdetector.

The invention also provides methods in which the analysis of biologicalsamples is a step in methods of diagnosis, treatment, or monitoring ofpatients suffering from a disease.

These methods can use the ablation cell, ablation apparatus, ICP ionsource or system as disclosed herein.

The invention also provides a method of labelling a biological samplewith four or more (e.g. 5, 6, 7, 8, 9, 10, 12, 13, 14, 15 or more)different transition metal atoms. Thus the samples in the methodsdiscussed above may be labelled with four or more (e.g. 5, 6, 7, 8, 9,10, 12, 13, 14, 15 or more) different transition metal atoms. Theseatoms associate with specific cellular targets, thereby permittingdownstream detection by LA-ICP-MS. Data from the LA-ICP-MS can then beused to create an image of the sample.

The invention also provides a method of imaging a biological sample,wherein multiple cells of the sample are subjected to mass cytometry ata subcellular resolution.

The effectiveness of these techniques is demonstrated in reference 7, asalso discussed below. This document describes mass cytometry using rareearth metals on antibodies to label target proteins in a biologicalsample. The sample is then analysed by an LA-ICP-MS system such as thatdescribed herein, and the simultaneous imaging of 32 proteins wasdemonstrated.

LA-ICP-MS and Mass Cytometry

The invention uses laser ablation coupled to inductively coupled plasmamass spectrometry (LA-ICP-MS) in a method of imaging a biologicalsample, such as a tissue sample. Different target molecules in thesample are labelled with different labelling atoms and LA-ICP-MS is thenused across multiple cells of the labelled sample. By linking detectedsignals to the known positions of the laser ablations which gave rise tothose signals the method permits localisation of the labelled targetmolecule to specific locations on the sample, and thus construction ofan image of the sample.

LA-ICP-MS involves subjecting the sample to laser pulses which generateplumes of ablated material from the sample, and these plumes aretransferred as aerosols to an ICP-MS instrument for analysis. Thelabelling atoms in the sample can be distinguished by MS and so theirdetection reveals the presence or absence of multiple targets in aplume.

The spatial resolution of signals generated in this way depends on twomain factors: (i) the spot size of the laser, as signal is integratedover the total area which is ablated; and (ii) the speed at which aplume can be analysed, relative to the speed at which plumes are beinggenerated, to avoid overlap of signal from consecutive plumes asmentioned above.

Thus, in order to analyse individual cells the invention uses a laserspot size which is no larger than these cells, and more specificallyuses a laser spot size which can ablate material with a subcellularresolution. This size will depend on the particular cells in a sample,but in general the laser spot will have a diameter of less than 4 μme.g. within the range 0.2-4 μm, 0.25-3 μm, or 0.4-2 μm. Thus a laserspot can have a diameter of about 3 μm, 2 μm, 1 μm, or 0.5 μm. In apreferred embodiment the laser spot diameter is within the range of0.5-1.5 μm, or about 1 μm. Small spot sizes can be achieved usingdemagnification of wider laser beams and near-field optics. A laser spotdiameter of 1 μm corresponds to a laser focus point of 1 μm, but thelaser focus point can vary by +20% due to numerical aperture of theobjective that transfers the laser beam onto the sample surface.

For rapid analysis of a sample a high frequency of ablation is needed,for example 10 Hz or more (i.e. 10 ablations per second, giving 10plumes per second). In a preferred embodiment the frequency of ablationis within the range 10-200 Hz, within the range 15-100 Hz, or within therange 20-50 Hz. An ablation frequency of at least 20 Hz allows imagingof typical tissue samples to be achieved in a reasonable time. As notedabove, at these frequencies the instrumentation must be able to analysethe ablated material rapidly enough to avoid substantial signal overlapbetween consecutive ablations. It is preferred that the overlap betweensignals originating from consecutive plumes is <10% in intensity, morepreferably <5%, and ideally <1%. The time required for analysis of aplume will depend on the washout time of the ablation cell, the transittime of the plume aerosol to and through the ICP, and the time taken toanalyse the ionised material.

Thus an ablation cell with a short washout time (e.g. 100 ms or less) isadvantageous for use with the invention. One example of such an ablationcell is the ablation cell discussed hereinabove. A cell with a longwashout time will either limit the speed at which an image can begenerated or will lead to overlap between signals originating fromconsecutive sample spots (e.g. reference 8, which had signal duration ofover 10 seconds). Therefore aerosol washout time is a key limitingfactor for achieving high resolution without increasing total scan time.Ablation cells with washout times of <100 ms are known in the art. Forexample, reference 9 discloses an ablation cell with a washout timebelow 100 ms. A particularly suitable ablation cell was disclosed inreference 10 (see also reference 11) which has a washout time of 30 msor less, thereby permitting a high ablation frequency (e.g. between20-40 Hz) and thus rapid analysis. The examples herein demonstrate thatthe signals of plumes generated by 20 Hz laser ablation shots can befully separated by this ablation cell, thereby enabling imaging of alarge area in short time. Using methods of the invention, it istherefore possible to achieve a time per spatially resolved pixel in afinal image of less than 100 ms.

The transit time of a plume aerosol to and through the ICP is easilycontrolled simply by positioning the ablation cell near to the ICP andby ensuring a sufficient gas flow to transport the aerosol at anappropriate speed directly to the ICP. Transport using argon and heliumas described in reference 10 provides good results.

The time taken to analyse the ionised material will depend on the typeof mass analyser which is used for detection of ions. For example,instruments which use Faraday cups are generally too slow for analysingrapid signals. Overall, the desired imaging speed (and thus ablationfrequency), resolution (and thus laser spot size and ablation cell) anddegree of multiplexing will dictate the type(s) of mass analyser whichshould be used (or, conversely, the choice of mass analyser willdetermine the speed, resolution and multiplexing which can be achieved).

Mass spectrometry instruments that detect ions at only onemass-to-charge ratio (m/Q, commonly referred to as m/z in MS) at a time,for example using a point ion detector, will give poor results in singlecell imaging using multiple labels. Firstly, the time taken to switchbetween mass-to-charge ratios limits the speed at which multiple signalscan be determined, and secondly, if ions are at low abundance thensignals can be missed when the instrument is focused on othermass-to-charge ratios. Thus, although the instrument used in references4 and 5 (Agilent 4500) is sensitive, its quadrupole-based detector isnot well suited to imaging with multiple labels because, by design, ionsof different mass-to-charge ratios pass through sequentially and so dataacquisition for multiple labels is slow. Similarly, the instruments usedin references 6 and 10 (Thermo Fisher ElementXR and Element2) analyseonly one m/Q at a time and have a large settling time for magnet jumpswhen measuring multiple m/Q values over a range exceeding the range ofan electrostatic field jump.

Thus it is preferred to use a technique which offers substantiallysimultaneous detection of ions having different m/Q values. Forinstance, instead of using a point ion detector, it is possible to usean array detector (e.g. see Chapter 29 of ref 12). Multi-collectorsector field ICP-MS instruments can be used (e.g. the Thermo ScientificNeptune Plus, Nu Plasma II, and Nu Plasma 1700 systems), and inparticular those having a Mattauch-Herzog geometry (e.g. the SPECTRO MS,which can simultaneously record all elements from lithium to uranium ina single measurement using a semiconductor direct charge detector).These instruments can measure multiple m/Q signals substantiallysimultaneously. Their sensitivity can be increased by including electronmultipliers in the detectors. Array sector instruments are not ideal,however, because, although they are useful for detecting increasingsignals, they are less useful when signal levels are decreasing, and sothey are not well suited in situations where labels are present athighly variable concentrations.

The most preferred MS method for use with the invention is based ontime-of-flight (TOF) detection, which can quasi-simultaneously registermultiple masses in a single sample. In theory TOF techniques are notideally suited to ICP ion sources because of their space chargecharacteristics, but it has been shown that TOF instruments can analysean ICP ion aerosol rapidly enough and sensitively enough to permitfeasible single-cell imaging (e.g. reference 7). Whereas TOF massanalyzers are normally unpopular for atomic analysis because of thecompromises required to deal with the effects of space charge in the TOFaccelerator and flight tube, imaging methods of the invention can beeffective by detecting only the labelling atoms, and so other atoms(e.g. those having an atomic mass below 100) can be removed. Thisresults in a less dense ion beam, enriched in the masses in (forexample) the 100-250 dalton region, which can be manipulated and focusedmore efficiently, thereby facilitating TOF detection and takingadvantage of the high spectral scan rate of TOF. Thus rapid imaging canbe achieved by combining TOF detection with choosing labelling atomsthat are uncommon in the sample and ideally having masses above themasses seen in an unlabelled sample e.g. by using the higher masstransition elements. Using a narrower window of label masses thus meansthat TOF detection to be used for efficient imaging.

Suitable TOF instruments are available from Tofwerk, GBC ScientificEquipment (e.g. the Optimass 9500 ICP-TOFMS), and Fluidigm Canada (e.g.the CyTOF™ and CyTOF™ 2 instruments). These CyTOF™ instruments havegreater sensitivity than the Tofwerk and GBC instruments and are knownfor use in mass cytometry because they can rapidly and sensitivelydetect ions in the mass range of rare earth metals (particularly in them/Q range of 100-200) [13]. Thus these are preferred instruments for usewith the invention, and they can be used for imaging with the instrumentsettings already known in the art e.g. references 14 & 15. Their massanalysers can detect a large number of markers quasi-simultaneously at ahigh mass-spectrum acquisition frequency on the timescale ofhigh-frequency laser ablation [6]. They can measure the abundance oflabelling atoms with a detection limit of about 100 per cell, permittingsensitive construction of an image of the sample. Because of thesefeatures, mass cytometry can now be used to meet the sensitivity andmultiplexing needs for imaging at subcellular resolution. Previously,mass cytometry has been used only to analyze cells in suspension, andinformation on cell-cell interactions within or tumor microenvironmentshas therefore been lost. By combining the mass cytometry instrument witha high-resolution laser ablation system and a rapid-transitlow-dispersion ablation chamber it has been possible to permitconstruction of an image of the sample with high multiplexing on apractical timescale. Further details on mass cytometry can be found inreferences 3 and 16.

The choice of wavelength and power of the laser used for ablation of thesample can follow normal usage in cellular analysis by ICP-MS. The lasermust have sufficient fluence to cause ablation to a desired depth,without substantially ablating the supporting sample holder. A laserfluence of between 2-5 J/cm² at 20 Hz is typically suitable e.g. from3-4 J/cm² or about 3.5 J/cm². Ideally a single laser pulse will besufficient to ablate cellular material for analysis, such that the laserpulse frequency matches the frequency with which ablation plumes aregenerated. Lasers will usually be excimer or exciplex lasers. Suitableresults can be obtained using an argon fluoride laser (λ=193 nm). Usingan aperture of 25 μm this laser can be imaged by 25-fold demagnificationonto the samples to give a spot size with a 1 μm diameter. Pulsedurations of 10-15 ns with these lasers can achieve adequate ablation.Femtosecond lasers (i.e. with pulse durations <1 ps) can also be used,and would be beneficial due to reduced heat transfer into the sample,but they are very expensive and good imaging results can be achievedwithout them.

Overall, the laser pulse frequency and strength are selected incombination with the response characteristics of the MS detector topermit distinction of individual laser ablation plumes. In combinationwith using a small laser spot and an ablation cell having a shortwashout time, rapid and high resolution imaging is now feasible.

Thus the invention provides an LA-ICP-MS system in which the washouttime of the ablation cell is less than 100 ms. In some embodiments, thewashout time may be less than 90 ms, less than 80 ms, less than 70 ms,less than 60 ms, less than 50 ms, less than 40 ms or less than 30 ms. Insome embodiments, the mass analyser in the LA-ICP-MS system is a TOFmass analyser. In some embodiments, the LA-ICP-MS system comprises abiological sample.

The invention also provides an LA-ICP-MS system comprising:

(i) a biological sample wherein at least three different targetmolecules in the biological sample have been labelled with at leastthree different labelling atoms, and

(ii) a laser which has been adapted to ablate the biological sample witha spot size of less than 4 μm.

Constructing an Image

LA-ICP-MS can provide signals for multiple labelling atoms in plumes.Detection of a label in a plume reveals the presence of its cognatetarget at the position of oblation. By generating a series of plumes atknown spatial locations on the sample's surface the MS signals revealthe location of the labels on the sample, and so the signals can be usedto construct an image of the sample. By labelling multiple targets withdistinguishable labels it is possible to associate the location oflabelling atoms with the location of cognate targets, so the inventioncan build complex images, reaching levels of multiplexing which farexceed those achievable using existing techniques. Images generated bythe methods of the invention can reproduce the staining patterns and theproportion of cells expressing a given marker as determined by IFM,thereby confirming the invention's suitability for imaging (see e.g.reference 7).

Ideally the image will be constructed by performing raster scanning ofthe laser over the sample. The spacing of consecutive ablations in theraster scan (step size), and between adjacent lines in the raster scan,is ideally the same as the laser spot size which is used (e.g. 1 μmspacing for a 1 μm laser spot) in order to achieve complete coverage ofa region of interest. In some embodiments, however, methods can use astep size which is smaller than the laser spot size (e.g. at least 2×,4×, or 5× smaller) as this can lead to smaller ablation areas and thusimprove imaging resolution. For achieving the scanning it is possible tomove the laser, but it is usually more convenient to move the ablationcell (or the contents of the cell). The movement speed will depend onthe ablation frequency and the raster spacing e.g. with 1 μm rasterspacing and 20 Hz ablation the ablation cell will have a translationspeed of 20 μm/s. Support stages which can achieve step sizes of 1 μm ofless are available e.g. with 500 nm or 200 nm step sizes (or even less).

Methods of the invention are generally used to create two-dimensional(2D) images of a sample, based on the contents of an ablated surfacelayer. 3D images of a tissue can be prepared by assembling stacks of 2Dimages (in a x,y plane) from sections of a single sample which areadjacent in the z-axis. As an alternative to assembling 2D images inthis way, however, methods of the invention can also be used for direct3D imaging. This can be achieved in various ways. For instance, if theablation causes vaporisation with a substantially constant depth thenrepeated ablation at a single x,y point reveals progressively deeperinformation in the z-axis. If ablation does not have a substantiallyconstant depth then the volume of ablated material can be measured (e.g.relative to a standard of known volume), and this volume can be easilyconverted to a z-axis depth. Where 3D imaging is performed it ispossible to perform multiple z-axis ablations while x,y location ismaintained (‘drilling’), or to ablate a sample layer by layer (i.e.perform ablations of a x,y area before moving to a deeper z-axis layer).Layer-by-layer ablation is preferred. Accuracy of 3D imaging is limitedby factors such as re-deposition of ablated material, the ability tomaintain a constant ablation depth, and the ability of labels topenetrate into the sample, but useful results can still be achievedwithin the boundaries of these limitations.

Assembly of signals into an image will use a computer and can beachieved using known techniques and software packages. For instance,reference 5 used the GRAPHIS package from Kylebank Software, but otherpackages such as TERAPLOT can also be used. Imaging using MS data fromtechniques such as MALDI-MSI is known in the art e.g. reference 17discloses the ‘MSiReader’ interface to view and analyze MS imaging fileson a Matlab platform, and reference 18 discloses two softwareinstruments for rapid data exploration and visualization of both 2D and3D MSI data sets in full spatial and spectral resolution e.g. the‘Datacube Explorer’ program.

Images obtained using methods of the invention can be further analysede.g. in the same way that IHC results are analysed. For instance, theimages can be used for delineating cell sub-populations within a sample,and can provide information useful for clinical diagnosis. Similarly,SPADE analysis can be used to extract a cellular hierarchy from thehigh-dimensional cytometry data which methods of the invention provide[19].

Labelling of the Biological Sample

The invention provides images of samples which have been labelled with aplurality of different labelling atoms, wherein the labelling atoms aredetected in laser-ablated plumes by ICP-MS. The reference to a pluralityof different atoms means that more than one atomic species is used tolabel the sample. These atomic species can be distinguished using ICP-MS(e.g. they have different m/Q ratios), such that the presence of twodifferent labelling atoms within a plume gives rise to two different MSsignals.

The invention is suitable for the simultaneous detection of many morethan two different labelling atoms, permitting multiplex label detectione.g. at least 3, 4, 5, 10, 20, 30, 32, 40, 50 or even 100 differentlabelling atoms. Labelling atoms can also be used in a combinatorialmanner to even further increase the number of distinguishable labels.The examples demonstrate the use of 32 different labelling atoms in animaging method, but LA-ICP-MS is intrinsically suitable for paralleldetection of higher numbers of different atoms e.g. even over 100different atomic species [13]. By labelling different targets withdifferent labelling atoms it is possible to determine the cellularlocation of multiple targets in a single image.

Labelling atoms that can be used with the invention include any speciesthat are detectable by LA-ICP-MS and that are substantially absent fromthe unlabelled sample. Thus, for instance, ¹²C atoms would be unsuitableas labelling atoms because they are naturally abundant, whereas ¹¹Ccould in theory be used because it is an artificial isotope which doesnot occur naturally. In preferred embodiments, however, the labellingatoms are transition metals, such as the rare earth metals (the 15lanthanides, plus scandium and yttrium). These 17 elements provide manydifferent isotopes which can be easily distinguished by ICP-MS. A widevariety of these elements are available in the form of enriched isotopese.g. samarium has 6 stable isotopes, and neodymium has 7 stableisotopes, all of which are available in enriched form. The 15 lanthanideelements provide at least 37 isotopes that have non-redundantly uniquemasses. Examples of elements that are suitable for use as labellingatoms include Lanthanum (La), Cerium (Ce), Praseodymium (Pr), Neodymium(Nd), Promethium (Pm), Samarium (Sm), Europium (Eu), Gadolinium, (Gd),Terbium (Tb), Dysprosium (Dy), Holmium (Ho), Erbium (Er), Thulium (Tm),Ytterbium (Yb), Lutetium (Lu), Scandium (Sc), and Yttrium (Y). Forexample, the invention can use any of the isotopes of the lanthanides aslisted in Tables 1 to 5. In addition to rare earth metals, other metalatoms are suitable for detection by ICP-MS e.g. gold (Au), platinum(Pt), iridium (Ir), rhodium (Rh), bismuth (Bi), etc. The use ofradioactive isotopes is not preferred as they are less convenient tohandle and are unstable e.g. Pm is not a preferred labelling atom amongthe lanthanides.

In order to facilitate TOF analysis (see above) it is helpful to uselabelling atoms with an atomic mass within the range 80-250 e.g. withinthe range 80-210, or within the range 100-200. This range includes allof the lanthanides, but excludes Sc and Y. The range of 100-200 permitsa theoretical 101-plex analysis by using different labelling atoms,while permitting the invention to take advantage of the high spectralscan rate of TOF MS. As mentioned above, by choosing labelling atomswhose masses lie in a window above those seen in an unlabelled sample(e.g. within the range of 100-200), TOF detection can be used to providerapid imaging at biologically significant levels.

Labelling the biological sample generally requires that the labellingatoms are attached to one member of a specific binding pair (sbp). Thislabelled sbp is contacted with a sample such that it can interact withthe other member of the sbp (the target sbp member) if it is present,thereby localising the labelling atom to a specific location in thesample. The sbp that delivers the label to the target molecule is alsoreferred to herein as a specific labelling construct. The method of theinvention then detects the presence of the labelling atom at thisspecific location and translates this information into an image in whichthe target sbp member is present at that location. Rare earth metals andother labelling atoms can be conjugated to sbp members by knowntechniques e.g. reference 20 describes the attachment of lanthanideatoms to oligonucleotide probes for ICP-MS detection, reference 21describes the use of ruthenium to label oligonucleotides, and FluidigmCanada sells the MaxPar™ metal labelling kits which can be used toconjugate over 30 different labelling atoms to proteins (includingantibodies).

Various numbers of labelling atoms can be attached to a single sbpmember, and greater sensitivity can be achieved when more labellingatoms are attached to any sbp member. For example greater than 10, 20,30, 40, 50, 60, 70, 80, 90 or 100 labelling atoms can be attached to asbp member. For example, monodisperse polymers containing multiplemonomer units may be used, each containing a chelator such as DTPA.DTPA, for example, binds 3+ lanthanide ions with a dissociation constantof around 10⁻⁶ M [3]. These polymers can terminate in a thiol-reactivegroup (e.g. maleimide) which can be used for attaching to a sbp member.For example the thiol-reactive group may bind to the Fc region of anantibody. Other functional groups can also be used for conjugation ofthese polymers e.g. amine-reactive groups such as N-hydroxy succinimideesters, or groups reactive against carboxyls or against an antibody'sglycosylation. Any number of polymers may bind to each sbp member.Specific examples of polymers that may be used include straight-chain(“X8”) polymers or third-generation dendritic (“DN3”) polymers, bothavailable as MaxPar™ reagents. Use of metal nanoparticles can also beused to increase the number of atoms in a label.

As mentioned above, labelling atoms are attached to a sbp member, andthis labelled sbp member is contacted with the sample where it can findthe target sbp member (if present), thereby forming a labelled sbp. Thelabelled sbp member can comprise any chemical structure that is suitablefor attaching to a labelling atom and then for imaging according to theinvention.

In general terms, methods of the invention can be based on any sbp whichis already known for use in determining the location of target moleculesin samples (e.g. as used in IHC or fluorescence in situ hybridisation,FISH), but the sbp member which is contacted with the sample will carrya labelling atom which is detectable by ICP-MS. Thus the invention canreadily be implemented by using available IHC and FISH reagents, merelyby modifying the labels which have previously been used e.g. to modify aFISH probe to carry a label which can be detected by ICP-MS.

The sbp may comprise any of the following: a nucleic acid duplex; anantibody/antigen complex; a receptor/ligand pair; or an aptamer/targetpair. Thus a labelling atom can be attached to a nucleic acid probewhich is then contacted with a sample so that the probe can hybridise tocomplementary nucleic acid(s) therein e.g. to form a DNA/DNA duplex, aDNA/RNA duplex, or a RNA/RNA duplex. Similarly, a labelling atom can beattached to an antibody which is then contacted with a sample so that itcan bind to its antigen. A labelling atom can be attached to a ligandwhich is then contacted with a sample so that it can bind to itsreceptor. A labelling atom can be attached to an aptamer ligand which isthen contacted with a sample so that it can bind to its target. Thuslabelled sbp members can be used to detect a variety of targets in asample, including DNA sequences, RNA sequences, proteins, sugars,lipids, or metabolites.

In a typical embodiment of the invention the labelled sbp member is anantibody. Labelling of the antibody can be achieved through conjugationof one or more labelling atom binding molecules to the antibody, forexample using the MaxPar™ conjugation kit as described above. Antibodieswhich recognise cellular proteins that are useful for imaging arealready widely available for IHC usage, and by using labelling atomsinstead of current labelling techniques (e.g. fluorescence) these knownantibodies can be readily adapted for use in methods of the invention,but with the benefit of increasing multiplexing capability. Antibodiesused with the invention can recognise targets on the cell surface ortargets within a cell. Antibodies can recognise a variety of targetse.g. they can specifically recognise individual proteins, or canrecognise multiple related proteins which share common epitopes, or canrecognise specific post-translational modifications on proteins (e.g. todistinguish between tyrosine and phospho-tyrosine on a protein ofinterest, to distinguish between lysine and acetyl-lysine, to detectubiquitination, etc.). After binding to its target, labelling atom(s)conjugated to an antibody can be detected to reveal the location of thattarget in a sample.

The labelled sbp member will usually interact directly with a target sbpmember in the sample. In some embodiments, however, it is possible forthe labelled sbp member to interact with a target sbp member indirectlye.g. a primary antibody may bind to the target sbp member, and alabelled secondary antibody can then bind to the primary antibody, inthe manner of a sandwich assay. Usually, however, the invention relieson direct interactions, as this can be achieved more easily and permitshigher multiplexing. In both cases, however, a sample is contacted witha sbp member which can bind to a target sbp member in the sample, and ata later stage label attached to the target sbp member is detected.

One feature of the invention is its ability to detect multiple (e.g. 10or more, and even up to 100 or more) different target sbp members in asample e.g. to detect multiple different proteins and/or multipledifferent nucleic acid sequences. To permit differential detection ofthese target sbp members their respective sbp members should carrydifferent labelling atoms such that their signals can be distinguishedby ICP-MS. For instance, where ten different proteins are beingdetected, ten different antibodies (each specific for a different targetprotein) can be used, each of which carries a unique label, such thatsignals from the different antibodies can be distinguished. In someembodiments, it is desirable to use multiple different antibodiesagainst a single target e.g. which recognise different epitopes on thesame protein. Thus a method may use more antibodies than targets due toredundancy of this type. In general, however, the invention will use aplurality of different labelling atoms to detect a plurality ofdifferent targets.

If more than one labelled antibody is used with the invention, it ispreferable that the antibodies should have similar affinities for theirrespective antigens, as this helps to ensure that the relationshipbetween the quantity of labelling atoms detected by LA-ICP-MS and theabundance of the target antigen in the sample will be more consistentacross different sbp's (particularly at high scanning frequencies).

If a target sbp member is located intracellularly, it will typically benecessary to permeabilize cell membranes before or during contacting ofthe sample with the labels. For example when the target is a DNAsequence but the labelled sbp member cannot penetrate the membranes oflive cells, the cells of the sample can be fixed and permeabilised. Thelabelled sbp member can then enter the cell and form a sbp with thetarget sbp member. In this respect, known protocols for use with IHC andFISH can be utilised.

Usually, a method of the invention will detect at least oneintracellular target and at least one cell surface target. In someembodiments, however, the invention can be used to detect a plurality ofcell surface targets while ignoring intracellular targets. Overall, thechoice of targets will be determined by the information which is desiredfrom the method, as the invention will provide an image of the locationsof the chosen targets in the sample.

Typically, where at least one of a plurality of different targetmolecules in the tissue sample is labelled with at least one differentlabelling atom, each different target is labelled with a differentlabelling atom, so that the presence of absence of each target can bedetermined on the basis of the presence of absence of a specificlabelling atom. In some instances, it however, multiple targets may belabelled with the same atom. In the instance of a protein, this may bebecause the multiple targets labelled with the same atom share a commonepitope, meaning that the different proteins are bound by the sameantibody. One example of such a situation would be the labelling of allIgG molecules with an antibody which binds to the Fc region of theantibody, even though the IgG molecules may have variable domains ofdifference sequence and bind to a range of antigens. Alternatively, aprotein bound by an antibody may be a subunit of a range of differentmultimer complexes, meaning that each complex containing the subunitwill be labelled by the same antibody. An example of this is found inantibodies which bind to certain multimeric cytokines or receptors, forexample the p40 subunit is part of both the IL-12 cytokine and the IL-23cytokine. In the case of a nucleic acid, the same principle could applywhere various nucleic acid sequences share a common motif. One exampleof this situation is a nucleic acid which binds to Alu repeats.

Biological Samples

The invention provides methods and laser ablation cells, ion sources,ablation apparatus and systems for imaging a biological sample. In someinstances, this sample is a tissue sample. The tissue sample comprises aplurality of interacting cells, and the method subjects a plurality ofthese cells to laser ablation in order to provide an image of thesecells in the tissue sample. In general, the invention can be used toanalyse tissue samples which are now studied by IHC techniques, but withthe use of labels which are suitable for detection by LA-ICP-MS.

Any suitable tissue sample can be used in the methods described herein.For example, the tissue can be epithelium tissue, muscle tissue, nervetissue, etc., and combinations thereof. For diagnostic or prognosticpurposes the tissue can be from a tumor. In some embodiments a samplemay be from a known tissue, but it might be unknown whether the samplecontains tumor cells. Imaging can reveal the presence of targets whichindicate the presence of a tumor, thus facilitating diagnosis. Thetissue sample may comprise breast cancer tissue, for example humanbreast cancer tissue or human mammary epithelial cells (HMLE). Thetissue sample may comprise formalin-fixed, paraffin-embedded (FFPE)tissue. The tissues can be obtained from any living multicellularorganism, but will usually be human.

The tissue sample will usually be a section e.g. having a thicknesswithin the range of 2-10 μm, such as between 4-6 μm. Techniques forpreparing such sections are well known from the field of IHC e.g. usingmicrotomes, including dehydration steps, including embedding, etc. Thusa tissue may be chemically fixed and then sections can be prepared inthe desired plane. Cryosectioning or laser capture microdissection canalso be used for preparing tissue samples. Samples may be permeabilisede.g. to permit of reagents for labelling of intracellular targets (seeabove).

The size of a tissue sample to be analysed will be similar to currentIHC methods, although the maximum size will be dictated by the laserablation apparatus, and in particular by the size of sample which canfit into its ablation cell. A size of up to 5 mm×5 mm is typical, butsmaller samples (e.g. 1 mm×1 mm) are also useful (these dimensions referto the size of the section, not its thickness).

In addition to being useful for imaging tissue samples, the inventioncan instead be used for imaging of cellular samples such as monolayersof adherent cells or of cells which are immobilised on a solid surface(as in conventional immunocytochemistry). These embodiments areparticularly useful for the analysis of adherent cells that cannot beeasily solubilized for cell-suspension mass cytometry. Thus, as well asbeing useful for enhancing current immunohistochemical analysis, theinvention can be used to enhance immunocytochemistry. The invention canalso be used for imaging biofilms. The analysis of biofilms is importantin a medical setting because biofilms of infective reagents can form onmucous membranes in the body, or, for example, on indwelling catheters.

After being prepared, the sample will be placed into a laser ablationcell and then subjected to analysis according to the invention.

Combinations with Other Techniques

The method disclosed herein can also be used in techniques fordiagnosing and treating diseases, by characterising samples frompatients. In essence, the method can be used for any purpose for whichIHC is used currently (as known in the art, and summarised briefly, forexample, in reference 22). Thus the invention provides a method ofdiagnosing a disease in a patient comprising:

(i) determining the quantity, presence and/or location of at least oneof a plurality of target molecules in a biological sample from saidpatient, wherein each of the at least one target molecules has beenlabelled with an atom which can be distinguished by LA-ICP-MS toidentify each of the at least one different target molecules, byperforming LC-ICP-MS on the tissue sample to produce an image of thesample on which the quantity, presence and/or location of the at leastone of a plurality of target molecules is provided; and

(ii) comparing the image obtained in step (i) to images obtained from asubject known to be suffering from a disease, thereby diagnosing thedisease in the patient.

It is then possible to administer a treatment to the patient that isused to treat the disease identified in step (ii).

The invention also provides a method of monitoring the effectiveness ofa treatment in a patient comprising:

(i) determining the quantity, presence and/or location of at least oneof a plurality of target molecules in a biological sample from saidpatient, wherein each of the at least one target molecules has beenlabelled with an atom which can be distinguished by LA-ICP-MS toidentify each of the at least one different target molecules, byperforming LC-ICP-MS on the tissue sample to produce an image of thesample on which the quantity, presence and/or location of the at leastone of a plurality of target molecules is provided;

(ii) comparing the image obtained in step (i) to images of the tissuepreviously obtained from the patient, thereby determining whether thequantity, presence and/or location of the least one of a plurality oftarget molecules has been modulated by the treatment, and correlatingthis modulation with the effectiveness or ineffectiveness of thetreatment.

Based on this, it is then possible to decide whether to continue or toalter the treatment based on whether the treatment is effective againstthe disease.

The specific procedure used in step (i) of these methods can be any ofthe specific LC-ICP-MS methods discussed herein. By comparing the imageis meant that the LC-ICP-MS image from the subject known to be sufferingfrom a disease (i.e. what would commonly be called a reference standard)is analysed for the quantity, presence and/or location of at least oneof a plurality of target molecules and then the image generated in step(i) of the method is analysed in the same way. If the quantity, presenceand/or location of the marker(s) under analysis is seen to be similarbetween the patient and the reference standard from the subject, it isindicative that the patient may be suffering from the same disease asthe patient (e.g. cancer). This diagnosis thereby permits an appropriatetreatment strategy to be decided upon for the patient.

Diagnostic, monitoring and treatment aspects of the invention relate tothe following conditions and processes:

cancers such as leukemia, acute leukemia, acute lymphoblastic leukemia(ALL), acute lymphocytic leukemia, B-cell, T-cell or F AB ALL, acutemyeloid leukemia (AML), acute myelogenous leukemia, chromic myelocyticleukemia (CML), chronic lymphocytic leukemia (CLL), hairy cellleukemia,myelodyplastic syndrome (MDS), a lymphoma, Hodgkin's disease, amalignamt lymphoma, nonhodgkin's lymphoma, Burkitt's lymphoma, multiplemyeloma, Kaposi's sarcoma, colorectal carcinoma, pancreatic carcinoma,nasopharyngeal carcinoma, malignant histiocytosis, paraneoplasticsyndrome/hypercalcemia of malignancy, solid tumors, bladder cancer,breast cancer, colorectal cancer, endometiral cancer, head cancer, neckcancer, hereditary nonpolyposis cancer, Hodgkin's lymphoma, livercancer, lung cancer, non-small cell lung cancer, ovarian cancer,pancreatic cancer, prostate cancer, renal cell carcinoma, testicularcancer, adenocarcinomas, sarcomas, malignant melanoma, hemangioma,metastatic disease, cancer related bone resorption, cancer related bonepain, and the like, in particular, breast and prostate cancer; diagnosisof tumors of uncertain histogensesis (e.g. metastatic tumors from anunknown primary tumour);

diseases characterised by the build-up of amyloid plaques, such asAlzheimer's disease or Parkinson's disease;

confirmation of infectious agents in a biological sample;

precise diagnosis of different forms of muscle dystrophy and the like.

EXAMPLES

i. Experimental

Manufacture of Laser Ablation Cell

A cell top 10 was manufactured from a rectangular cuboid of acrylicglass (poly (methyl methacrylate), PMMA). A longitudinal hole of 3 mminner diameter was drilled through the cuboid along the long axis,forming the flow channel 11. In the top wall portion 17 of the cell top10, a transversal, slightly elliptically shaped hole with length L=4.5mm and a width of 1.5 mm was formed and was closed by an UV transparentsilica window 16. A lateral opening 14 of similar dimensions as the holeon the top side was formed in the bottom wall portion 15 of the cell top10. The bottom wall portion 15 was then machined to reduce the minimumthickness w of the bottom wall portion in the region of the flow channel11 to approximately 50 micrometers. The total length of the flow channel11 was about 50 mm.

A cell bottom 10 was manufactured from another PMMA cuboid. Acylindrical sample chamber 21 having a diameter of approximately 23 mmwas milled into the cuboid. A radially extending hole was drilled intothe cell bottom to form a sheath gas inlet 22. The sheath gas inletextended at an angle of 10° relative to the flow channel 11. A sample 23was placed in the sample chamber 21. The cell bottom 20 was mounted tothe cell top 10 with the aid of four screws (not shown in the drawings).No spacer or seal was required, but a spacer or seal may optionally beprovided to better seal of the contact region between the cell top 10and the cell bottom 20. The top surface 24 of the sample 23 faced thelateral opening 14. The distance d between the top surface 24 of thesample 23 and the bottom wall portion 15 was about 350 micrometers.Thus, the total distance between the sample surface 24 and the centeraxis of the flow channel 11 was approximately 1.9 millimeters (radius ofthe flow channel=1.50 mm, wall thickness w=0.05 mm, distance d=0.35 mm).

Experimental Set-Up for Tube Cell Performance, Optimization andCharacterization

An ArF excimer laser system (Lambda Physik, Göttingen, Germany) withhomogenized laser beam profile was coupled to an Agilent 7500csICP-Quadrupole-MS instrument (ICP-Q-MS, Agilent Technologies, Waldbronn,Germany). Laser fluence was 17.3 J/cm². In order to improve theconfidence level, all data points were derived from 3×3 single shotmatrix scans (unless otherwise stated) with laser spot size of 10 μm andspacing of 15 μm between adjacent shots. The transfer tube to the ICPtorch consisted of a 3 mm inner diameter PTFE (polytetrafluoroethylene)tubing connected to the mixed-gas outlet 13. A similar tube was used asa feed tube to the Ar inlet 12. The Ar carrier gas flow was adjusted to1.1 L/min. The 50 cm long transfer tube was directly connected to theICP torch without changing the diameter. The He sheath gas flow providedthrough the sample chamber was adjusted to 0.6 L/min. Tube cellperformance measurements were carried out using a dwell time of 10 ms.In order to describe the washout of the cell, single isotope ²⁷Alacquisitions during 1 Hz, 10 Hz and 30 Hz laser ablations on NIST 610reference glass were performed. The ICP was operated at 1470 W and thequadrupole MS was set to 1 point per peak in peak hopping mode.

Optimization of various operational parameters was carried out,including: the gap distance between carrier tube opening and the samplesurface; the Ar carrier gas flow rate; and the He sheath gas flow rate.When optimizing one parameter, the other two were set to the‘pre-optimized’ conditions, based on the preliminary optimization, e.g.gap distance at 350 μm, Ar flow at 1.1 L/min, He flow at 0.6 L/min. Thedata were evaluated based on the normalized peak width, which is thepeak width divided by the total counts collected within each peak (peakarea). For each peak, full width at 1% maximum (FW0.01M) was used todetermine the peak width. In case the 1% maximum position did notcoincide with any data point, a linear interpolation of the nearest twopoints was applied. For the peak area, all data points within the peakwidth were integrated and no interpolation was required, since peaktailings contributed to less than 1% of the total counts.

The characterization of the cell for routine analysis was carried outusing the same parameters as described above. However, multiple isotopesfrom low, mid to high m/Q were recorded in different runs. Peak areasensitivities were abundance corrected.

Sample Preparation for Imaging of “Hard” Matter

A sample for demonstrating imaging capabilities was produced by alaser-induced forward transfer method. For the preparation of the donorsubstrate, a high quality fused silica glass was covered with aUV-absorbing triazene polymer (TP) layer, as a sacrificial dynamicrelease layer (DRL), on which different thin film materials weredeposited. In the transfer procedure, a 308 nm XeCl excimer laser beamwas imaged on an ‘ETH’ (or ‘PSI’) hollow mask and 4 fold demagnifiedbefore impacting the back side of the donor substrate. TP-DRL wasablated and the generated shockwave propelling the thin film toward aglass receiver substrate coated with PEDOT:PSS(poly(3,4-ethylenedioxythiophene) blended with poly(styrenesulfonate)).The sample was prepared by a 60 nm thick Au ‘ETH’ thin layer on bottomand an 80 nm Ag ‘PSI’ on top (Au/Ag). To control the deposition of thetwo logos by scanning electron microscope (SEM), a 5 nm Pt thin film wasuniformly coated on the receiver substrate after the pattern deposition.

Tissue Sample Preparation

A formalin-fixed paraffin-embedded human epidermal growth factorreceptor 2 (HER2)-enriched breast cancer tissue was sectioned at 6 μm.The sample was processed on the Discovery XT platform (Ventana MedicalSystems) under CC1m epitope recovery conditions. Afterwards, the samplewas blocked for 30 minutes with phosphate buffered saline (PBS)/1%bovine serum albumin (BSA)/0.1% Triton X, and incubated with 200 μL¹⁶⁵Ho tagged anti-HER2 at 5 μg/mL for 50 minutes. The sample was washedthree times in PBS/0.1% Triton X and dried at room temperature. Forantibody conjugation with ¹⁶⁵Ho, a commercial MAXPAR antibody labelingkit (DVS Sciences) was employed.

Instrumentation and Operating Conditions for Imaging of “Hard” Matter

A similar configuration as described for the tube cell characterizationwas used for the experiments. An area of 852×408 μm² was covered by 10Hz repetition rate line scans. The distance between successive lasershots and the lateral distance between line scans were both 4 μm, basedon a 4 μm laser crater. The actual laser beam size was 1-2 μm. However,a larger affected area was observed, which can be explained by anenlarged heat penetration volume (high thermal diffusion in the metallicthin films, and ns light-material interaction time). Three isotopes,¹⁰⁷Ag, ¹⁹⁵Pt and ¹⁹⁷Au, were measured in peak hopping mode with 600 μsdwell time for each isotope. However, due to an instrumentquadrupole-settling time of a few milliseconds for each isotope, thereading of an entire set of isotopes could not be completed in less than10 ms. Obviously, such a large overhead fraction (low duty cycle) doeslimit signal quality obtainable during fast, high resolution imagingexperiments. Data analysis was based on the integration of each singleshot signal (trapezium integration scheme).

Instrumentation and Operating Conditions for Tissue Imaging

Tissue imaging was conducted on an Element2 (Thermo Fisher Scientific)ICPMS coupled to an ArF excimer laser at −1 μm spatial resolution. Theoperating conditions were optimized for maximum sensitivity of fasttransient signals. Therefore, only ¹⁶⁵Ho was recorded. For imageanalysis, the sample was scanned line by line at a laser frequency of 20Hz and an image pixel size of 1×1 μm². Dwell time of the MS was set to50 ms, in accordance with the laser ablation rate applied.

B. Results and Discussion

Tube Cell Optimization

The dependence of the dispersion on the gap between the tube cell bottomand the sample surface is depicted in FIG. 6 (a). The plotted peakwidths were normalized to the total counts collected in FW0.01M. Aminimum peak width at a gap width of ˜350 μm was observed. Using thisoptimized gap distance, the Ar and He gas flow rate optimizations werecarried out. The corresponding results are shown in FIG. 6 (b) and (c)based on the normalized peak widths. All three optimizations yield anevident minimum of ˜10⁻³ ms/counts for the normalized peak width. Allmeasurements reported in the following sections were conducted using theoptimized values for sample gap and gas flow rates. Depending ondifferent setups, such optimization values may vary.

Experimental Evaluation of the Tube Cell

The tube cell was characterized using a laser frequency of ˜1 Hz.Typical transient signals (shown in log-scale) are summarized in FIG. 7(a). A single washout signal of a major element lasted around 30 ms forFW0.01M. The transient peak has still a slightly asymmetric shape,tailing slightly which is caused by delayed washout of the aerosol.After the peak maxima, signals dropped down for more than 2 orders ofmagnitude within 20 ms, which represents more than 99.98% of the totalintegrated signal. The residual fraction of the total signal (0.02%integrated signal area) was found in the tail of the peak which reachedbackground after 40-50 ms. The slope of the second signal decay isdifferent than for the fast washout, indicating a different process,which is suspected to be related to the uptake of redeposited materialfrom the surface. This is most likely due to the flow of the He sheathgas into the tube cell bottom opening, which is parallel to the laserplume injection, which flushes the area around the crater mostefficiently. Spacing the single shots by 1 s blank indicates that nofurther sample removal occurs.

FIG. 7 (b) shows the transient signal acquired at a laser frequency of10 Hz. The peak width and shape were similar to those signals measuredat 1 Hz. Further increase in the laser frequency using a 30 Hz line scanis shown in FIG. 7 (c). The width and shape of the peaks were similar tothe signals measured at 1 Hz and 10 Hz. The signal structure indicatesthat two adjacent peaks cannot be separated to background from eachother. However, the overlapping between two successive peaks is lessthan 1% in intensity. Therefore it can be concluded that even 30 Hzwould allow to image concentration differences as large as two orders ofmagnitude, which makes this ablation cell very attractive for laserablation imaging. The entire evaluation demonstrates that the washout issignificantly improved into the 30-50 ms time range.

Tube Cell Characterization for Broad Isotope Range

Further characterizations of the tube cell performance are documented inFIG. 8. Peak widths and sensitivities calculated from peak areas areshown for different isotopes from low m/Q (⁷Li) up to high m/Q (²³⁸U).As seen from FIG. 8 (a), the mean peak widths of all the isotopemeasurements fall into a narrow range of 30-35 ms. The reported signaldurations were calculated based on FW0.01M. The standard deviationsacross the m/Q range are most likely the result of the ablated mass,aliasing effects, and fluctuations due to differences in the gas flowdynamics. FIG. 8 (b) shows furthermore the normalized sensitivities forthe peak area, which were determined using 10 μm craters in single shotablation mode. Compared to commonly used ablation cell setups in singleshot mode, the peak area sensitivities improved by a factor of 10. Thisis not related to improved sample transport efficiency or improvedionization and purely based on the preserved sample density from theablation site to the ICP.

Fast Imaging by Sequential Q-MS

The “hard” sample was studied by various imaging techniques. The resultsare illustrated in FIG. 9. The characteristic details of the patternwere imaged first by optical microscopy (FIG. 9 (a)) and scanningelectron microscopy (SEM, FIG. 9 (b)). These images were used toevaluate the quality of the high sensitivity, high spatial resolutionLA-ICPMS for ¹⁹⁷Au (FIG. 9 (c)) and ¹⁰⁷Ag (FIG. 9 (d)). The optical andSEM images indicate that the thin film patterns were not perfect interms of homogeneity, shape and geometry. However, the sample wasconsidered to be well suited to be analyzed by LA-ICPMS. LA-ICPMSproduced highly consistent images with sharp pattern boundaries. Therapid signal change from the thin film to background (or backwards) wasconsidered as an indicator for high spatial resolution of about 1 μm. Ascratch was introduced to the left arm of ‘T’ during sampletransportation from one to the other laboratory and even this was imagedby LA-ICPMS in FIG. 9 (c) and is consistent with the optical microscopeimage in FIG. 9 (a) taken as a control picture.

Fast Imaging by Simultaneous Mattauch-Herzog Mass Spectrometer

A severe limitation of standard Quadrupole mass spectrometers is theirsequential m/Q analyzing scheme. The short signal pulse durationresulting from the low dispersion tube cell limits the recording ofmultiple isotopes, unless quasi- or simultaneous mass spectrometers arecoupled to LA-ICP. Examples of such advanced MS include Mattauch-HerzogMS (MH-MS) and Time-of-Flight-MS (TOF-MS). In order to illustrate themulti-element imaging capabilities of a MH-MS instrument, the sameLA-ICP system as described for quadrupole ICPMS was coupled. The imagesobtained with this system were of similar quality and resolution as forquadrupole MS. It should be mentioned here that an LA-ICP-TOF-MScoupling would be equally suited for such rapid chemical imagingapplications.

Tissue Imaging

Among the many possible applications of the presently disclosedelemental imaging LA-ICPMS system, it was decided to demonstrate itspotential by investigating biomarker distributions in a biologicaltissue thin section. Such analyses demand, first, low m resolution toresolve the morphology of and to localize biomarkers within the smallestbiological unit, the cell. This information is crucial in the study ofbiological processes and for comprehensive diagnostic purposes. Second,such analyses demand a short measurement time per pixel, as feasibleusing the presently disclosed ablation cell; in biological andbiomedical analyses typically a large number of samples and large tissueareas (500×500 μm²) need to be analyzed for statistical purposes.Therefore, a breast cancer tissue section was analyzed to investigatethe human epidermal growth factor receptor 2 (HER2) statuses ofindividual cells illustrated in FIG. 6. The image showed HER2 proteinhighly expressed on the cell membrane. HER2 is a major determinant ofrelapse free survival time, time to metastasis and overall survival timeafter an initial breast cancer diagnosis. In the analysis, ˜1 μm spatialresolution was achieved. Such high spatial resolution and chemicalsensitivity allowed a highly precise HER2 determination in the breastcancer tissue. This sub-cellular resolution of an important biomarkerfor breast cancer analysis may be suitable to guide pathologists intheir various treatment options.

It will be understood that the invention is described above by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

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What is claimed is:
 1. A laser ablation inductively coupled plasma massspectrometry (LA-ICP-MS) system for imaging of biological samples,comprising: a laser ablation cell comprising a flow channel and a samplechamber; wherein the flow channel extends from a carrier gas inlet to amixed gas outlet; wherein the flow channel comprises a lateral openingand a lateral window; wherein the sample chamber comprises a sheath gasinlet; a laser adapted to shoot a laser beam through both the lateralwindow and the lateral opening of the ablation cell and impinge on thesurface of a sample received by the sample chamber to generate a plumefrom the sample; an inductively coupled plasma (ICP) torch coupled to anoutlet of the ablation cell for receiving and ionizing the plume; and amass analyzer coupled to the ICP torch.
 2. The system of claim 1,wherein the carrier gas inlet is outside the sample chamber.
 3. Thesystem of claim 1, wherein carrier gas flowing through the carrier gasinlet does not pass through the sample chamber.
 4. The system of claim1, wherein the system is arranged to deliver Argon into the carrier gasinlet and Helium into the sheath gas inlet.
 5. The system of claim 3,wherein the system is arranged to deliver Argon into the carrier gasinlet and Helium into the sheath gas inlet.
 6. The system of claim 1,wherein the lateral opening but not the lateral window is adjacent tothe sample chamber.
 7. The system of claim 1, wherein the laser ablationcell is adapted to have a washout time of 100 ms or less.
 8. The systemof claim 1, wherein the flow channel is in a cell top of the laserablation cell.
 9. The system of claim 8, wherein the cell top isdetachable from the sample chamber.
 10. The system of claim 1, whereinthe system further comprises a positioning device operable to change therelative position between a sample received by the sample chamber andthe laser beam.
 11. The system of claim 10, wherein the positioningdevice is operable to scan the laser beam over the sample surface. 12.The system of claim 1, wherein the lateral opening has a cross sectionalarea that is not more than 20 mm².
 13. The system of claim 1, whereinthe laser is a UV laser.
 14. The system of claim 1, wherein the laser isoperable to shoot a pulsed laser beam, and wherein each laser pulse willgenerate the plume from the sample in the sample chamber.
 15. The systemof claim 1, wherein the laser has a laser spot size of 4 μm or less. 16.The system of claim 1, wherein the mass analyzer is a time-of-flightmass analyzer.
 17. The system of claim 1, wherein the sample chambercomprises a biological sample comprising a plurality of different targetmolecules each labeled with an antibody attached to a lanthanidelabeling atom.
 18. The system of claim 17, wherein the biological samplecomprises at least 10 different labeling atoms.
 19. A laser ablationinductively coupled plasma mass spectrometry (LA-ICP-MS) system forimaging of biological samples, comprising: a laser ablation cellcomprising a flow channel and a sample chamber; wherein the flow channelextends from a carrier gas inlet to a mixed gas outlet; wherein thecarrier gas inlet is outside the sample chamber; wherein the flowchannel comprises a lateral opening and a lateral window; wherein thesample chamber comprises a sheath gas inlet; a laser adapted to shoot alaser beam through both the lateral window and the lateral opening ofthe ablation cell and impinge on the surface of a sample received by thesample chamber to generate a plume from the sample; an inductivelycoupled plasma (ICP) torch coupled to an outlet of the ablation cell forreceiving and ionizing the plume; and a mass analyzer coupled to the ICPtorch.
 20. A laser ablation inductively coupled plasma mass spectrometry(LA-ICP-MS) system for imaging of biological samples, comprising: alaser ablation cell comprising a flow channel and a sample chamber;wherein the flow channel extends from a carrier gas inlet to a mixed gasoutlet; wherein the flow channel comprises a lateral opening and alateral window; wherein the sample chamber comprises a sheath gas inlet;wherein carrier gas flowing through the carrier gas inlet does not passthrough the sample chamber; a laser adapted to shoot a laser beamthrough both the lateral window and the lateral opening of the ablationcell and impinge on the surface of a sample received by the samplechamber to generate a plume from the sample; an inductively coupledplasma (ICP) torch coupled to an outlet of the ablation cell forreceiving and ionizing the sample; and a mass analyzer coupled to theICP torch.